Tea Is The Second Most Popular

Published Date: 02 Nov 2017

CHAPTER 1

INTRODUCTION

Tea is the second most popular non-alcoholic drink in the world (Sharma. et al., 2007). The leaves and buds of tea plant Camellia sinensis undergo withering, steaming, rolling and dried to produce tea (Ho, Lin and Shahidi, 2009). It is native to the southern provinces of China and parts of India, Laos, Thailand, Vietnam and Myanmar (Cabrera, Artacho and Gimenez, 2006).

Teas can be categorized into four major types based on the fermentation level. Green and white teas are non-fermented, oolong tea is semi-fermented, and black tea and red tea (Pu-Erh) are fermented (Cabrera, Artacho and Gimenez, 2006). Green tea and white tea are produced by drying and steaming the fresh leaves without prior subjection to fermentation (Cabrera, Artacho and Gimenez, 2006). Alternatively, the steps involved to produce oolong tea are partial fermentation, drying and steaming (Cabrera, Artacho and Gimenez, 2006). Fresh tea leaves are subjected to post-harvest fermentation before drying and steaming to produce black and red teas (Cabrera, Artacho and Gimenez, 2006). Polyphenol oxidase and microorganisms are used to catalyze the oxidation in fermentation stage for black tea and red tea respectively (Cabrera, Artacho and Gimenez, 2006).

Green tea consists of proteins, amino acids such as glycine, tryptophan tyrosine and valine, carbohydrates such as cellulose, glucose, fructose and sucrose, lipids such as linoleic and linolenic acid, sterols such as stigmasterol vitamins B.C and E, chlorophyll, carotenoids, minerals, trace elements such as Ca, Mg, Mn, Se and Na, aldehydes, alcohols, esters, lactones, hydrocarbons, xanthic bases such as caffeine and theophylline, polyphenols and flavonoids (Cabrera, Artacho and Gimenez, 2006).

The common forms of green tea available in the market are tea bag, loose tea and ready-to-drink tea products (Squidoo, 2011). Tea bag can be brew in hot or cold (Squidoo, 2011). It is preferred by people with busy life style as it can be prepared easily and sipped slowly (Squidoo, 2011). Alternatively, people who enjoy to hand-make their tea bags or prepare tea using a filtering device prefer loose tea (Squidoo, 2011). Particularly, youth in the world preferred ready-to-drink tea product for instance canned or bottle tea drinks (Ho, Lin and Shahidi, 2009).

On the other hand, liquid green tea concentrate, green tea extract in capsule and green tea tablets are the less likely forms of green tea that can be found in the tea industry (Squidoo, 2011). Green tea extract in capsule or tablets act as supplement product which provide adequate of tea polyphenols and methylxanthine needed per day (Squidoo, 2011). In contrast, liquid green tea concentrate allow for rapid preparation and direct benefit from the tea components such as theobromine, vitamins and minerals (Squidoo, 2011).

The appropriate amount of tea components for instance catechins and caffeine can bring beneficial effect to health. However, toxicity effect such as liver failure, dizziness and death can happen if those tea components are taken in excess. Moreover, the amount of the respective tea components required depends on the age, gender and health conditions.

The average amount of caffeine required by healthy adult is 200 to 300 mg/day which is equal to two to three cups of tea (Mayo Clinic, 2011). Excess amount of caffeine lead to osteoporosis as it will increase the amount of calcium in urine and sweat (MedlinePlus, 2011). Besides, it can also cause "caffeine jitters" such as sleepless, diarrhea and irritability (About.com, 2012). Health Canada recommended that the daily amount of caffeine intake for adolescence is 2.5 mg/kg body weight (Health Canada, 2011). Alternatively, the caffeine intake for pregnant and breast-feeding women should not be more than 200 mg/day as excess amount of caffeine will cause miscarriage and some other negative effects (MedlinePlus, 2011). The risk of Parkison’s disease can be decreased by consuming 421-2710 mg/day which equals to 5 to 33 cups of green tea for men while women need 1 to 4 cups of green tea per day (MedlinePlus, 2011). Furthermore, the cholesterol level can be decreased by consuming more than 10 cups of tea per day (MedlinePlus, 2011).

The proper dosage of catechins required is 300 to 700 mg/day which is equal to 3 to 6 cups of tea per day (Amazing Green Tea, n.d.). In addition, consumption of 200 to 300 mg/day of EGCG helps in maintaining cardiovascular and metabolic health (American College of Nutrition, 2006).

Additionally, green tea polyphenols able to provide some health benefits which include antioxidant, antimutagenic, anticarcinogenic, antihypertensive and cardiovascular disease risk, oral health and solar ultraviolet protection (Cabrera, Artacho and Ggimenez, 2006).

On the other hand, different analytical techniques are developed and used to measure tea polyphenols. The most common techniques that used to identify the polyphenolic compounds are paper chromatography (PC) (Zuo et al., 2002, p.308), thin layer chromatography (TLC) (Zuo et al., 2002, p.308), column chromatography (Yao et al., n.d.) and colorimetric technique (Yao et al., n.d.). These techniques are linked to redox potential (Yao et al., n.d.). Colorimeter techniques and measurement of radical scavenging activity are indirect approaches that used to estimate the total polyphenolic content of tea. These methods are non-specific but it is very helpful to quantify the non-characterized polyphenols in tea (Beecher et al., 1999). Alternatively, gas liquid chromatography (GC) (Zuo et al., 2002, p.308) and high performance liquid chromatography (HPLC) (Zuo et al., 2002, p.308) are the advance techniques that used to isolate, identify and quantify individual polyphenolic compounds.

Matsumoto (1997) suggested that commercial green teas consist of more polyphenols as compared to other types of tea. Even so, limited researches are reported about the polyphenol content in green tea done in Malaysia. Therefore, this research is conducted to understand and set baseline information on polyphenolic content in commercial green teas obtained from different geographic origins and available in Malaysian market.

Due to its varied health benefits and popularity, the present study is aimed with the following objectives:

1) To isolate and quantify the green tea polyphenols using reverse phase-HPLC with UV detection

2) To determine the total phenolic content and total flavanoid content of commercial green tea using UV-Vis spectrophotometer

3) To compare the polyphenols present in commercial green tea from different countries

4) To learn the proper techniques of HPLC and spectrophotometer

CHAPTER 2

LITERATURE REVIEW

2.1 Background of Tea

Tea was first discovered in 2737 B.C. by Chinese Emperor, Shan Nong. Shan Nong is a renowned herbalist (United Kingdom Tea Council, n.d.) had a practice of boiling his drinking water (Wissotzky Tea, 2007). One day a few leaves from a tree were sudden fell into his boiling water and a rich and attractive aroma was emitted (Wissotzky Tea, 2007). The tree was a Camellia sinensis and the drink was known as tea (United Kingdom Tea Council, n.d.). He found out that the drink was refreshing and energizing (Wissotzky Tea, 2007). Therefore, he had instructed to plant the tea brushes in the gardens of his palace and this brewing practice began and rapidly spread (Wissotzky Tea, 2007).

On 5th century A.D., the China’s upper class adopted the trend of presenting packages of tea as gift. Moreover, they also retain the practice of drinking tea at social events and in private homes (Wissotzky Tea, 2007). Around the same time, Japanese Buddhist monks who study at China had introduced tea to Japan and tea drinking became part of the Japanese culture (United Kingdom Tea Council, n.d.). On the other hand, tea was introduces to Europe in 16th century by Portuguese who stay in East as traders and missionaries. However, Dutch were the first instead of Portuguese who ship back tea as commercial import (United Kingdom Tea Council, n.d.). Tea started to spread from Dutch to other countries in Western Europe but it only practices by Europe’s upper class due to its high price (United Kingdom Tea Council, n.d.). At the early of 18th century, the price of tea consequent decreases with the increase of tea imports (Wissotzky Tea, 2007). This enable the tea develop into a common product which can enjoyed by all sectors of the population (Wissotzky Tea, 2007).

2.2 Tea Plants description

Tea plant is the plant under Camellia family (The Fragrant Leaf, n.d.). It is native to China, Tibet and northern India (The Fragrant Leaf, n.d.). In addition, it can classify into Camellia sinenesis also known as Chinese plant and Camellia assamica also known as Assam plant (Donhauser, 2006 cited in Belde, 2011). Camellia sinensis is small leaf variety which able to grow in the cold and high regions of central in China and Japan (The Fragrant Leaf, n.d.). Alternatively, Camellia assamica is a broad leaf variety which thrives in moist and tropical climate found in Northeast India and Szechuan and Yunnan provinces of China (The Fragrant Leaf, n.d.).

The tea quality was depends on the environment conditions and processing techniques. High quality of tea often produced from tea plants which grow in higher mountain and hand plucking. Mountain conditions are more favorable for the growth of tea plants while hand plucking able to produce highly uniformity (Ho, Lin and Shahidi, 2009).

2.3 Chemical composition of tea

Tea consists mainly of polyphenolic catechins, theaflavins and alkaloids. All of these components are secondary metabolites of tea plant which has defense properties to protect the plant against pathogens (Friedman. et al., 2005).

Epigallocatechin-3-gallate, epigallocatechin, epicatechin-3-gallate and epicatechin are the primary polyphenolic catechins which are present mainly in green tea and white tea. On the other hand, black tea consists mostly of theaflavins which include theaflavin, theaflavin-3-gallate and theaflavin-3, 3-digallate. The alkaloids: caffeine and theobromine are major components in all types of tea (Friedman. et al., 2005).

Figure 1: Structures of tea components: Catechins, theaflavins and alkaloids (Friedman. et al., 2005).

In addition, green tea also composes of gallic acid, flavonols such as quercetin, myricetin and kaempferol and some other phenolic compounds for instance chlorogenic acid and caffeic acid (Cabrera, Artacho and Ggimenez, 2006).

Figure 2: Chemical structure of caffeine and theophylline

(Cabrera, Artacho and Ggimenez, 2006).

Alternatively, green tea using old leaves have highest amount of EGCG and total catechins follow by green tea using young leaves and oolong tea (Cabrera, Artacho and Ggimenez, 2006). Black tea and Pu-Erh have the lowest level of EGCG and total catechins (Cabrera, Artacho and Ggimenez, 2006).

The factors that affect the relative catechin content of green tea are geographic, growing conditions such as soil, climate agriculture practice and fertilizers, the type of green tea, preparation of infusion and the way the leaves been processed before drying (Cabrera, Artacho and Ggimenez, 2006).

2.4 Consumption pattern of tea among world population

Varieties of commercial teas such as white, green, black, oolong and pu-erh tea were found in the form of loose tea leaves, tea cakes or tea bags (Xiaohun Wan et al. 2009). Different consumption pattern can found in different parts of the world and yet some of the countries do not or prefer less to classify tea as one of their beverages. On the other hands, loose tea leaves are more likely to consume by countries with high tea production like China and Indonesia (theworld’s healthiest foods, n.d.). Meanwhile, hot infusion of green tea also known as unfermented fresh green shoots are  mainly consumed by far east countries especially China, Japan and Korea while fermented tea or black tea is a main beverage in most of the countries. Besides, the serving pattern of green tea is much limited as compared to black tea and usually it is serves without adding sugar or milk. Most of the Islāmic countries such as Libya and Malaysia prefer to add sugar, cream or milk into the brewed black tea which can found either in the form of loose tea leave or tea bags. For instance European countries like France and Germany favor more to have sweet tea instead of bitter tea while the majority of the European nations prefer to have bitter tea (Lipton, n.d.). Ready-to-drink tea products such as bottled or canned tea are preferred by all youths the world alike (Wan, Li and Zhang, n.d. cited in Ho, Lin and Shahidi, 2009).

2.5 Beneficial effects of Green tea

Cholesterol. Lin et al. (1998) reported that the prolong consumption able to lowers the total cholesterol and increase the HDL content in both animals as well as people. Tea polyphenols able to decrease the risk of atherosclerosis and cardiovascular disease by prevent the oxidation of LDL (Singh et al., 2002). In addition, green tea polyphenols might play a role to inhibit the absorption of cholesterol via intestine and enhance excretion from the body (Anderson 2011). Thus, the lipid absorption in the body decreased.

Brain Health. EGCG was the major component of green tea has the neuroprotective effects (Anderson 2011) against neurodegeneration diseases for instance Parkison’s and Alzheimer’s disease (Winred et al., 2004; Pan, Jankovic and Lee 2003). Lee et al. (2009) investigated that the treatment using EGCG capable to improved the memory function and restrain the fibrilization of Abeta in vitro. Hence, the development or progression of Alzheimer’s disease was inhibited.

Hypertension. It is interconnected with atherosclerosis, stroke, renal failure and cardiovascular disease (Hypertension control 1996). Yang et al. (2004) concluded that consistent consumption of 120 ml/day of green tea or oolong tea for more than a year, able to reduce the risk of developing hypertension in Chinese population.

Anticarcinogenic. Anderson (2011) reported that green tea catechins have the chemopreventive effects which depend on antioxidant action, development and apoptosis, molecular regulatory functions on cellular growth and specific induction of detoxifying enzymes. In addition, green tea polyphenols especially EGCG was the excellent growth inhibitor in most of the tested cell lines and it only act on cancer cells (Anderson 2011). Alternatively, EGCG induce the DNA destruction of cancer cells, apoptosis and cancer cell cycle arrest (Anderson 2011).

Weight Loss. According to Dulloo et al. (1999), weight loss directly related to the leptin level which monitor to the energy intake and energy expenditure through brain signals. Green tea polyphenol (EGCG) do play a role in reducing leptin and other hormone levels which vital in regulating appetite. In addition, tea polyphenols can increase the noradrenaline levels which play a role in activating the brown fat tissue that burns calories from the white fat located around human hips, thighs and waistline (Anderson 2011). Alternatively, the extract of green tea was important in control the body composition and weight maintenance by increase the energy expenditure which associated to fat oxidation (Dulloo et al., 1999).

Solar Ultraviolet Protection. Katiyar (2003) reported that green tea polyphenols especially EGCG have the photo-protective properties against some type of radiation that lead to skin disease, photoaging and potential cancer. Besides, the application of green tea polyphenols provides the protection against UVB-induced inflammation, oxidative stress as well as UVB-induced local and systemic immune suppression (Katiyar 2003).

Diabetes. Zhang and Kashket (1998) concluded that tea polyphenols have the ability to reduce serum glucose by prevent the amylase activity which can found in saliva and intestines. As a result, the increase in serum glucose can be minimized due to the decomposition of starch has been delay.

2.6 Tea Toxicity

Particular dose of tea and its constituents might give a number of beneficial effects on health. Beyond the moderate dose can cause adverse effects to health (Chacko, et al., 2010).  The reasons which cause the adverse effect of tea over consumption are caffeine content, presence of aluminum and the effect of tea polyphenols (Cabrera, Artacho and Ggimenez, 2006).  

According to Cabrera, "fermented" teas such as black tea and red tea consist of high amount of caffeine as compare to "non-fermented" teas such as green and white teas. Moreover, the caffeine content in teas can affected by the type of tea, the form of preparation and available forms of tea. In addition, bagged tea consist higher percentage of caffeine than tea leaves (Willson, 1999). High amount of caffeine can cause restlessness, nervousness, tachycardia (rapid heart rate), vomit, headaches, epigastric pain (Cabrera, Artacho and Ggimenez, 2006), deteriorate the diarrhea (WebMD, n.d.), frequent urination, loss of appetite (Amazing Green Tea, n.d.) and increase blood sugar level (Amazing Green Tea, n.d.). Alternatively, pregnant and breast-feeding women who consume more than two cups per day can increase the risk of miscarriage (WebMD, n.d.), increase the risk of neural tube birth defect in babies (Amazing Green Tea, n.d.) and lower birth weight (WebMD, n.d.).  Besides, it will cause the baby become more irritable and have more bowel movements (WebMD, n.d.). Overdose of caffeine also will lead to osteoporosis as it increases excretion of calcium via urination. Furthermore, it will affect the rate of blood clotting (WebMD, n.d.).  

Some studies shown that tea-plant have high-capacity to accumulate aluminum. Aluminum can accumulate by the body and dietary intake excess 6 mg/day may lead to osteomalacia and neurodegenerative disorders for instance Alzheimer’s disease (Cabrera, Artacho and Ggimenez, 2006).

In contrast, over dietary intake of Epigallocatechin Gallate (EGCG) which can found in green tea extract can cause cytotoxicity in liver (Chacko, et al., 2010) and neural tube defects (Wonder of Tea, n.d.). Besides, EGCG serve as pro-oxidant instead of antioxidant in pancrease β cells in vivo (Yun, Kim and Song, 2006). Moreover, EGCG mimic insulin, rise tyrosine phosphorylation of insulin receptor and insulin receptor substrate and diminish the gene expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (Chacko, et al., 2010). As a result, high intake of green tea might be unfavorable for diabetic animals to control hyperglycemia (Yun, Kim and Song, 2006). On the other hand, green tea catechins formed complex with iron which may inhibit bioavailability of non-heme iron from diet. In consequence, it will worsen anemia (Cabrera, Artacho and Ggimenez, 2006).

2.7 Methods used for Analysis

2.71 UV-Vis Spectrometric Technique

Spectrometric techniques are quantitative analyses (Andersen and Markham, 2006, p.115) that are widely used to determine the total polyphenols and total flavanoids content of various teas (Harbowy and Balentine, 1997; Lakenbrink. et al., 2000 cited in Yao.et al., n.d.).

2.72 Total polyphenolic content

Folin-ciocalteu (FC) assay is a quantitative assay, easy, reproducible and often applies in the routine of quality control and to measure the antioxidant capacity of food products as well as dietary supplements (Ainsworth and Gillespie, 2007). FC assay involve the spectroscopic determination of blue complexes which result from the transfer of electrons in alkaline medium from phenolic compound to phosphomolybdic or phosphotungstic acid complexes (Ainsworth and Gillespie, 2007) using specific wavelengths to determine polyphenols present in tea (Dunja. et al., 2008).

2.73 Total flavanoid content

Total flavonoid content was determined using aluminium chloride colorimetric assay (Jia. et al., 1999: Chang. et al., 2002 cited in Rosa, Parilla and Aguilar, 2010, p.141). However, not all the subgroups of flavanoids can identify using colorimetric method (Boyles. et al., 1993 cited in Rosa, Parilla and Aguilar, 2010, p.141). The principle is the colorimetric reagent (Aluminium chloride) from acid stable complexes with C-4 keto group and either the C-3 or C-5 hydroxyl group of flavones and flavonols (Chang, et al., 2002). Furthermore, it forms acid labile complexes with the orthodihydroxyl group in the A- or B-ring of flavonoids (Chang, et al., 2002).The range for maximum absorbance for the complexes formed by flavonols with C-3 and C-5 hydroxyl groups is 415-440 nm (Chang, et al., 2002). Quercetin is reported to be appropriate to build the calibration curve (Kiranmai, Kumar and Ibrahim, 2011).

2.74 Reverse-phase High Performance Chromatography

HPLC is one of the liquid chromatography (Waters: The Science of What’s Possible, 2012) which was use for the separation and identification of the compounds in the sample that can dissolve in a liquid such as water or ethanol (TheLinde Group, n.d.). It is very useful in determining the precise amount of individual polyphenols and flavanoids in tea (Beecher, Warden and Merken, 1999). The basis separations by HPLC are partition, adsorption, ion exchange and size exclusion (Sood, et al., 2009).

In addition, it can categorize into normal-phase HPLC and reverse phase HPLC (Tosoh Bioscience GmbH, n.d.). RP-HPLC used to separate non-polar and hydrophobic components in sample. It involves the hydrophobic interactions between sample and stationary phase (Tosoh Bioscience GmbH, n.d.). Column-18(C18) bound silica is commonly used as stationary phase in RP-HPLC (Waters: The Science of What’s Possible, 2012). C18 is hydrophobic, which able to bind to polar components in sample or standard, using polar solvent (Peptracker, n.d.). Normal phase-HPLC involve isolation of hydrophilic and polar components in the extract (Tosoh Bioscience GmbH, n.d.). The stationary phase involves is polar and non-polar mobile phase is used (Sood, et al., 2009).

RP-HPLC is select for this research as it is the most preferable, sensitive and reliable method that use to identify and quantify the respective polyphenols in tea (Lee and Ong, 2000; Longo. et al., n.d.). Lee and Ong (2000) as cited in Friedman (2005) reported that tea analysis using HPLC was five times more sensitive than electrophoresis. Besides, it enables precise control of variant, for instance, type of solvent, temperature, pH and concentration (Tosoh BioscienceGmbH, n.d.). It can also be applied to extensive range of molecule, which consists of polar and charged molecules (Tosoh Bioscience GmbH, n.d.).

There are two types of elution for RP-HPLC which are isocratic and gradient elution. Isocratic elution is the mobile phase composition that remains constant throughout the separation whereas gradient elution involves the change of mobile phase composition during the run (Waters: The Science of What’s Possible, n.d.).

Gradient elution of RP-HPLC is applied in this research to isolate tea component with different polarities (Jamdera, et al., 2005). The reason isocratic elution is not chosen is due to the fact that peaks produced for part of the less polar catechins will be broadening and tailing (Wang. et al., 2000; Lin. et al., 1998 cited in Zuo, Chen and Deng, 2002, p.308).   

On the other hand, the main detectors apply in HPLC analyses are UV detector, evaporating light scattering detector and fluorescence detector (Sood, et al., 2009). UV detector is choose for this study.

CHAPTER 3

MATERIALS AND METHODS

3.1 Sample selection

The samples were chosen based on the production of green tea at different geographical areas. The green teas selected do not contain any flavor compound. This is to prevent interference caused by flavor compounds. These samples were in tea bags form.

The selected commercial green teas were purchased from AEON Mid Valley (Selangor), Mercato Pavillion (Kuala Lumpur), Cold Storage Times Square (Kuala Lumpur) and Carrefour Mid Valley (Selangor).

Table 3.1: Brand name and manufacturing countries of commercial green tea

Brand name

Manufacturing Country

g/tea bag

A

Japanese Green Tea Sencha

Japan

2

B

Lipton: Clear Green

Indonesia

2

C

TenRenTea: Fresh Green Tea

Taiwan

4

D

TeaZen:First Flush Pure Green Tea

Korea

2

E

BOH: Green Tea

Malaysia

1.5

F

Dilmah: Special Green Tea

Sri Lanka

2

G

Duchy Originals from Waitrose: Organic Green Tea

China

2.5

H

Teekanne: Finest Green Tea

German

1.75

I

Ahmad Tea London: Organic Green tea

London

2

Note: samples A to G are categorized under tea produced in Asian countries; Sample H and I are categorized under tea produced in Europe countries.

3.2 Samples Preparation

Samples were extracted with deionized water to test the amount of tea polyphenols per serving. Each green tea bag with 1.5-4g was brewed in 250ml of deionized water at 80 °C for 10 minutes. The extracts were filtered through filter paper followed by 0.45µm nylon syringe filter.

The extracts were freshly prepared for HPLC analysis and UV-VIS spectrometry analysis. This was important to minimize oxidation of tea polyphenols as the polyphenols are very unstable and photosensitive (Lee and Ong, 2000).

3.3 HPLC analysis

3.3.1 Standard preparation

The stock solutions of epicatechin-3-gallate, theaflavin, theaflavin-3-gallate, catechin, gallic acid and caffeine were prepared by dissolving the respective standards in deionized water. Meanwhile, theobromine was dissolved in acidified water. The solutions were vortexed, sonicated and stored at -18°C before use. All the standards were protected from light to prevent degradation. Following tables shown the standards used as well as the concentrations of respective standards.

Table 3.3 (a): Purity and the name of the company for selected standard

Standard

Company

Purity %

Epicatechin-3-gallate

CalBiochem

98.1

Catechin

Sigma Life Science

98.0

Gallic acid

Merck Schuchardt OHG

99.0

Theobromine

Otganics

99.0

Caffeine

Merck KGaa Acros

98.5

Table 3(c): Different concentration of standard.

Standard

Concentration/mM

Epicatechin-3-gallate

0

0.1

0.2

0.3

Catechin

0

0.1

0.2

0.3

Gallic acid

0

0.1

0.2

0.3

Theobromine

0

0.1

0.2

0.3

Caffeine

0

0.2

0.4

0.6

3.32 Instrumentation

Shimadzu GCMS-QP 2010 Ultra equipped with an Autosampler, Shimadzu UELC CBM-20A was used to conduct RP-HPLC analysis for this research. The stainless-steel column-18 with 150 mm long and 4.6 mm inner diameter was packed with 5 µm of ZORBAX Rx-SIL particles as stationary phase. The temperature of the column was maintained at 38°C using Shimadzu column oven CTO-10AS VP. The mobile phases used for RP-HPLC analysis at a

pH 2.65 of acidified water as solvent A, which consists of acetic acid and deionized water as well as solvent B, which consists of 95 % acetonitrile. Both solvents were filtered through 0.45µm membrane before use. The flow rate applied was 0.8 ml/min for a total run of 30.10 minutes. The gradient condition for the complete 30.1-minute extraction was shown in table 3(d). The initial composition of the mobile phase consists of 95% of solvent A and 5% of solvent B. Next, Solvent B was gradually increased to 15% in 10 min, 45% in 15 min, 55% in 20 min, 60% in 30 min and return to 5% in 30.1 min. The peaks for samples and standards were detected using UV-VIS detector (Shimadzu SPD-20A UV-VIS) at 280 nm. 20 µl of tea extract was injected into the column for analysis. The analysis for each sample and standard were conducted in triplicate. The step of pre-column washing after an analysis was important to ensure that the remaining tea components are washed out. In addition, it also prevents the interference of remaining components with the tea components for the next analysis (Furusawa 2001).

The purpose of using acidified water as solvent A was to enhance the stability of catechin and other polyphenolic compounds (Zuo, Chen and Deng 2002). Besides, it also enables the elimination of peak tailing and allows the complete and efficient resolution of polyphenolic compounds in the teas (Beecher, Merken and Warden 1999). The pH of the acidified water does play important roles in separation of the components present in test samples. According to Jandera et al (2005), baseline noise, retention and separation increases as the pH apply is lower than the optimum pH. Besides, the pH apply is higher than optimum able to minimize the baseline noise and shorten the analysis time. However, the signal intensity decreases. As stated by Zuo et al (2002), 280nm was the excellent detection wavelength for catechins, gallic acid and caffeine in tea leaves.

Table 3(d): Mobile phase gradient flow ramp

Time (min)

Solvent A (%)

Solvent B (%)

0-0.01

95

5

0.01-10.00

85

15

10.00-15.00

55

45

15.00-20.00

45

55

20.00-30.00

40

60

30.00-30.10

95

5

3.33 Data Analysis

LC-Solution was the software that used to be identified the retention times and peak area for standards and samples. The phenolic components in tea samples were determined by comparing the retention times of each peak with retention times of the standards. The concentration of phenolic compounds in tested samples was determined with the used of calibration curves.

Calculation

3.3.4 Recovery test

It acts as a confirmation test for the peaks identified (Friedman et al., 2005). Besides, it is used for accuracy assessment to measure the effectiveness of sample preparation (LabCompliance, n.d.). In addition, the percentage of recovery indicates the purity of the compound (Stevens et al. 2009).

The green tea samples was spiked with known amount of respective standards before brewed in 250 ml deionized water at 80 °C for 10 minutes. The peak areas obtained were compared with the peak area for sample and standard.

3.4 Total Flavonoid Content Assay

3.41 Reagents

The reagents which applied in total flavonoids content assay are Quercetin hydrate from Acros Organics and aluminium chloride obtained from Merck Schuchardt OHG.

3.42 Instrumentation

Aluminum chloride colorimetric assay was used to determine the total flavanoid content in the selected samples (Baghiani, 2012). 0.5 ml of 2 % aluminum chloride was added to 0.25 ml of extracted sample and standard solution of Quecetin in 0.25 ml of distilled water. The mixture was mixed and incubated at room temperature for an hour. The absorbance was determined against the blank at 420 nm using Genesys 20 Thermo Scientific. Standard solution of Quercetin was prepared by dissolving Quercetin in 80 % ethanol and standard curve was plotted to determine the total flavanoid content in samples. 2% of aluminum chloride was prepared by dissolving 2 g of aluminum chloride anhydrous in 100 ml of 80 % ethanol. Blank solution consists of only 0.5 ml 80 % ethanol in 0.5 ml 2 % aluminum chloride.

Table 3(e): Concentration of Quercetin used.

Concentration of Quercetin/mM

0.10

0.12

0.14

0.16

0.18

0.20

Calculation

3.5 Folin-Ciocalteu Assay

3.51 Reagents

The reagents for Folin-ciocalteu assay were Folin-Ciocalteu’s phenol reagent attained from Sigma-Aldrich, Sodium carbonate anhydrous from systerm, gallic acid anhydrous obtained from Merck Schuchardt OHG.

3.52 Instrumentation

Total phenolic content was determined using Folin-Ciocalteau assay (Ainsworth and Gillespie, 2007). 45 µl of standard solution of Gallic acid or diluted sample was added to 155 µl of distilled water followed by 300 µl of 10 % Folin-Ciocalteau reagent (F-C reagent). 400 µl of 20 % sodium carbonate was added to the mixture after 30 seconds and mixed. The absorbance was measured after two hours of incubation at room temperature against the blank at 765 nm using Genesys 20 Thermo Scientific. The total phenolic content of samples was determined using standard curve. Blank solution consists of only distilled water, 10 % F-C reagent and 20 % sodium carbonate. 20 % sodium carbonate was prepared by boiling the mixture of 4 g anhydrous sodium carbonate and 16ml distilled water. A few crystals of sodium carbonate were added after the mixture was cooled to room temperature and stayed overnight for 24 hours. The mixture was filtered and topped up to 20 ml with water. Standard solution was prepared by dissolving anhydrous gallic acid in 1 ml of 95 % ethanol and 9 ml of water. 15 µl of extracted samples was diluted with 30µl of water before used in F-C assay.

Table 3(f): Concentration of Gallic acid used.

Concentration of Gallic acid/mM

0.5

1.0

1.5

2.0

Calculation

3.6 Statistical Analysis

Analysis for all samples and standards were carried out in triplicate. The statistical tests were performed using Microsoft Excel 2007. The mean, standard error and relative standard deviation were determined. ANOVA was used to determine the significance of the mean at P<0.05. Calibration curves plotted using Microsoft Excel were used to determine the concentration of total polyphenols content, total flavanoids content and respective polyphenols in selected commercial green teas.